Phylogenetic analysis of a dataset of fungal 5.8S rDNA sequences shows that highly divergent copies of internal transcribed spacers reported from Scutellospora castanea are of ascomycete origin

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.     

High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae)

Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same. Interestingly, the Mimulus 5.8S sequence was much more divergent than other higher-plant rDNA sequences. Consideration of the secondary structure of the 5.8S rRNA shows that most of the changes in Mimulus are compensatory and preserve the basic secondary structure of the mature RNA molecule.     

Extensive 5.8S nrDNA polymorphism in <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and identification of mycorrhizal fungi associated with <Emphasis Type="Italic">Stigmatodactylus sikokianus</Emphasis> (Maxim. ex Makino) Rauschert (Orchidaceae)

The mycorrhizal fungi of Stigmatodactylus sikokianus (Orchidaceae) were isolated and identified to be nearly related to Sebacina spp. in Sebacinaceae (Basidiomycota) by a neighbor-joining phylogenetic analysis based on the sequences of the ITS region of nuclear rDNA. In spite of the geographically separated samplings, high sequence similarity was found among the obtained DNA sequences, which suggested that S. sikokianus might be highly specialized to the group of fungi. It is known that Sebacina spp. are saprobes or ectomycorrhiza-forming fungi. The mycorrhizal fungi of S. sikokianus were regarded to be saprobic from the environment of their habitats.     

Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid <Emphasis Type="Italic">Aegilops</Emphasis> (L.) species

The nucleotide sequence of the fragment of the internal transcribed spacer (ITS) of rDNA comprising the full-length ITS1, the gene encoding 5.8S rRNA, and part of the ITS2 sequence was determined in 22 samples of five diploid Aegilops species. The full alignment length of compared sequences was 524 bp. Species-specific substitutions were found in the ITS nucleotide sequence of rDNA of different Aegilops species. Intraspecific differences in ITS structure in diploid Aegilops species were detected for the first time. Polymorphism of the ITS nucleotide sequence within the same sample was revealed, which might be due either to differences between the genomes of individual plants comprising the sample or to the presence of several types of ribosomal genes in the genome of one plant. In general, both interspecific and intraspecific variability of the ITS nucleotide sequences of rDNA is extremely low. In total, 26 variable sites, twelve of which were informative, were identified.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 193–197.Original Russian Text Copyright © 2005 by Goryunova, Chikida, Gori, Kochieva.     

Rhodotorula lamellibrachii sp. nov., a new yeast species from a tubeworm collected at the deep-sea floor in Sagami Bay and its phylogenetic analysis

A new species of the genus Rhodotorula was isolated from a tubeworm (Lamellibrachia sp.) collected at a depth of 1156 m in Sagami Bay, Japan. Strain SY-89 had physiological properties quite similar to R. aurantiaca. Two phylogenetic trees, one based on internal transcribed spacer (ITS) regions and 5.8S rDNA sequences and the other based on the D1/D2 region of the large subunit (26S) rDNA sequences, united strain SY-89 to the type strain of Sakaguchia dacryoides through a considerable evolutionary distance. Strain SY-89 was differentiated from S. dacryoides by the G+C content of the nuclear DNA and differences in the ability to utilize specific carbon and nitrogen compounds. The low complementarity of strain SY-89 DNA to that of the type strain of S. dacryoides confirmed that this strain was genetically unrelated to previously known species. The tubeworm isolates are described as R. lamellibrachii sp. nov. The type strain of R. lamellibrachii is strain SY-89 (= JCM 10907). R. lamellibrachii formed a cluster with Erythrobasidium hasegawianum, R. lactosa, S. dacryoides and Sporobolomyces elongatus on the ITS and 5.8S rDNA phylogenetic tree. These five species shared a signature sequence in 26S rDNA, although this relationship was not supported by phylogeny based on the D1/D2 region of 26S rDNA.     

Molecular phylogeny ofSalicaceae and closely relatedFlacourtiaceae: Evidence from 5.8 S, ITS 1 and ITS 2 of the rDNA

A ribosomal DNA region, including the entire 5.8S RNA gene and the internal transcribed spacers ITS 1 and ITS 2, was used for studying the phylogeny ofSalicaceae and the relationship betweenSalicaceae andFlacourtiaceae. The length of the ITS regions withinSalicaceae andFlacourtiaceae was similar to that found in other angiosperms. The GC content of both ITS regions was high, varying 62.7-72.2%. The most parsimonious tree clusters the wind-pollinatedChosenia bracteosa among theSalix species, suggesting that it should be included in the genusSalix. The grouping withinSalix leaves subg.Salix as paraphyletic, for which reason the subgeneric division is questionable.Populus was monophyletic and formed a sister group toSalix. The interspecific variation of the ITS sequences was very small inSalicaceae, which is in contradiction to the age of the group according to the evidence from fossil data.Idesia polycarpa fromFlacourtiaceae shows great sequence similarity withSalicaceae, but the analysis of 5.8S rDNA supports monophyly of the four species ofFlacourtiaceae sampled for this study.     

Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA

Summary Nucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.     

The sequence of the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway in <Emphasis Type="Italic">Penicillium</Emphasis> species

Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence of the isoepoxydon dehydrogenase (idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA (ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

四照花亚属的nrDNA ITS致同进化不完全

四照花亚属(Cornus subg.Syncarpea)隶属于山茱萸科山茱萸属(Cornus),我国该亚属共有5种8亚种。为探讨四照花亚属nrDNA ITS序列的致同进化不完全现象及假基因产生的可能原因,分析了该亚属4种(每种1~2个居群)共21个个体的nrDNA ITS序列。结果表明,这些类群的nrDNA ITS存在多态性,通过分析这些nrDNA ITS克隆序列的G+C含量、5.8S保守基序和二级结构最小自由能,推测其可能存在假基因。系统发育研究结果显示所有nrDNA ITS序列分成5个分支,同一个体的不同拷贝被分别置于两个甚至多个分支中,且不同分支显示了不同种间关系。四照花亚属物种个体内部存在nrDNA ITS不完全致同进化,可能归咎于不完全的世系分选(incomplete lineage sorting)、种间杂交或多倍化等进化事件,从而导致基因组内nrITS区序列出现多态性,同时也导致难以通过外部形态来划分亚属内种间界限。     

Nucleotide Sequences of Plasmodium vivaxA-Type rDNA ITS1, 5.8S,and ITS2. Elaboration of Retrospective PCR Diagnostics of Malaria Using Stained Thick Blood Smears

Stages life cycle of the malaria parasite differ in the rate of replication and the structural properties of functionally active A-, S-, and O-type ribosomes. Regions of A-type rDNA including ITS1, 5.8S, and ITS2 from two strains of Plasmodium vivaxwith different incubation periods were amplified and sequenced. No substantial differences in the sequences of two strains were revealed. Phylogenetic analysis of the obtained and homologous sequences of ITS1 rDNA of A, S, and O types of P. vivax; A and S types of P. falciparum; and Cryptosporidium parvum, Eimeria maxima, Toxoplasma gondiias outgroup, by the maximum parsimony method using PAUP 4.0 revealed that divergence of ITS1 might have occurred after speciation and at different rates in individual lineages of the Plasmodiumgenus. Basing on the results of the analysis of orthologous sequences of P. vivaxand P. falciparum, we developed genus- and species-specific primers for PCR diagnostics of malaria, as well as a one-step effective method of DNA isolation from Giemsa–Romanovsky-stained thick blood smears. It was demonstrated that stained preparations could be a reliable source of plasmodial DNA, and the quality of preparations and storage time (10–20 years) did not interfere with the results of PCR analysis.     

Improved phylogenetic resolution of toxic and non-toxic Alexandrium strains using a concatenated rDNA approach

Dinoflagellates of the genus Alexandrium are known producers of paralytic shellfish toxins. Species within the genus have similar phenotypes making morphological identification problematical. The use of Alexandrium rDNA sequence data is therefore increasing, resulting in the improved resolution of evolutionary relationships by phylogenetic inferences. However, the true branching pattern within Alexandrium remains unresolved, with minimal support shown for the main phylogentic branch. The aim of this study is to improve phylogenetic resolution via a concatenated rDNA approach with a broad sample of taxa, allowing inference of the evolutionary pattern between species and toxins. 27 Alexandrium strains from 10 species were tested with HPLC for PSP toxin presence and additionally sequenced for 18S, ITS1, 5.8S, ITS2 and 28S rDNA before being phylogenetically inferred together with all available orthologous sequences from NCBI. The resulting alignment is the largest to date for the genus, in terms of both inferred characters and taxa, thus allowing for the improved phylogenetic resolution of evolutionary patterns there in. No phylogenetic pattern between PSP producing and non-producing strains could be established, however the terminal tamarense complex was shown to produce more PSP analogues than basal clades. Additionally, we distinguish a high number of polymorphic regions between the two copies of A. fundyense rDNA, thus allowing us to demonstrate the presence of chimeric sequences within GenBank, as well as a possible over estimation of diversification within the tamarense complex.     

Molecular characterization and evaluation of mycorrhizal capacity of Suillus isolates from Central Spain for the selection of fungal inoculants

Suillus fungal specimens of pine forests from a Mediterranean area of central Spain (Madrid region) were studied based on molecular and physiological analysis of sporocarps to obtain fungal native inocula to produce mycorrhizal Pinus halepensis Miller in nursery. Variation within the internal transcribed spacer (ITS) region of the ribosomal RNA genes of Suillus was examined by restriction fragment length polymorphism (RFLP) and direct sequencing of polymerase chain reaction products. Ribosomal DNA (rDNA) spacers were amplified from pure cultures obtained from fruit bodies of a range of Suillus species: Suillus bellinii (Inzenga) Watling, Suillus bovinus (Pers.) Kuntze, Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.) Snell, Suillus mediterraneensis (Jacquet. & Blum) Redeuil, Suillus luteus L. (Gray), and Suillus variegatus (Sw.) Kuntze. Interspecific variation in the length and number of restriction sites of the amplified ITS region was observed. This variation was confirmed by sequencing, which allowed us to identify some isolates. This is the first time that the ITS sequence of S. mediterraneensis is completely described. No intraspecific rDNA variation was observed within isolates of S. collinitus, S. mediterraneensis, and S. luteus. The phylogenetic analysis established the close relationship among these Mediterranean fungal species. As a further step to characterize the different isolates and to understand the relation between genetic and functional diversity, some physiological variables were evaluated. Intraspecific variation in axenic fungal growth and in mycorrhizal capacities was detected, especially within S. collinitus isolates. The fungal isolates stimulated the growth of P. halepensis in different rates. These studies indicated that ITS analysis, in conjunction with mycorrhizal tests, provides suitable combined tools for the analysis of Suillus spp. in a small geographic area for selecting isolates with final afforestation purposes.     

Community of arbuscular mycorrhizal fungi in drought-resistant plants, <Emphasis Type="Italic">Moringa</Emphasis> spp., in semiarid regions in Madagascar and Uganda

The community structure of arbuscular mycorrhizal (AM) fungi in the roots of drought-resistant trees, Moringa spp., was examined in semiarid regions in Madagascar and Uganda. Root samples were collected from 8 individuals of M. hildebrandtii and 2 individuals of M. drouhardii in Madagascar and from 21 individuals of M. oleifera in Uganda. Total DNA was extracted from the root samples, and partial nSSU rDNA of AM fungi was amplified using a universal eukaryotic primer NS31 and an AM fungalspecific primer AM1. The PCR products were cloned and divided by restriction fragment length polymorphism (RFLP) analysis with HinfI and RsaI. Some representatives in each RFLP types were sequenced, and a neighbor-joining phylogenetic analysis was conducted for the obtained sequences with analogous sequences of AM fungi. The RFLP and phylogenetic analyses showed that AM fungi closely related to Glomus intraradices or G. sinuosum were detected in many samples. The AM fungal groups frequently detected in the Moringa spp. might be widely distributed species in semiarid environments.     

Mycorrhizal specificity, preference, and plasticity of six slipper orchids from South Western China

Mycorrhizal fungi of six endangered species, Paphiopedilum micranthum, Paphiopedilum armeniacum, Paphiopedilum dianthum, Cypripedium flavum, Cypripedium guttatum, and Cypripedium tibeticum, from two closely related genera in the Orchidaceae from Southwestern China, were characterized using the nuclear internal transcribed spacer (ITS) and part of the large subunit gene of mitochondrial rDNA (mtLSU) sequences. The most frequently detected fungi belonged to the Tulasnellaceae. These fungi were represented by 25 ITS sequence types and clustered into seven major clades in the phylogenetic analysis of 5.8S sequences. Species of Paphiopedilum and Cypripedium shared no fungal ITS sequence types in common, but their fungal taxa sometimes occurred in the same major clade of the 5.8S phylogenetic tree. Although it had several associated fungal ITS sequence types in a studied plot, each orchid species had in general only a single dominant type. The fungal sequence type spectra of different species of Paphiopedilum from similar habitats sometimes overlapped; however, the dominant sequence types differed among the species and so did the sequence-type spectra within Cypripedium. Orchids of P. micranthum and P. armeniacum transplanted from the field and grown in two greenhouses had a greater number of mycorrhizal associations than those sampled directly from the field. Root specimens from P. micranthum taken from the greenhouses were preferably associated with mycobionts of the Tulasnella calospora complex, while those from the field had mycorrhizal associations of other tulasnelloid taxa. Such plasticity in mycorrhizal associations makes ex situ conservation or even propagation by means of mycorrhization of axenically grown seedlings possible.     

Botryosphaeria spp. isolated from apple and several deciduous fruit trees are divided into three groups based on the production of warts on twigs, size of conidia, and nucleotide sequences of nuclear ribosomal DNA ITS regions

We examined the phytopathological and biological characters ofBotryosphaeria spp. isolated from apples and other deciduous fruit trees, and determined the nucleotide sequences of their rDNA ITS regions. TheBotryosphaeria isolates from deciduous fruit trees can be divided into three groups based on their production of warts on twigs, size of the conidia, and nucleotide sequences of rDNA ITS 1, ITS 2 and 5.8S rDNA. Isolates ofBotryosphaeria in ITS group A produced conidia of intermediate size and showed warts on infected twigs prior to the development of ring rot on fruit. This group was common on deciduous fruit trees in Japan as a causal agent of ring rot and wart bark diseases of apples and pears; and it appears similar to theB. dothidea from the US that was isolated from apple exhibiting white rot. The ITS group BBotryosphaeria produced small conidia and induced shoot blight without wart development prior to the development of ring rot on fruit. This group was localized on pear, persimmon, and kiwi fruit in restricted areas of Japan. The ITS group CBotryosphaeria consisted ofB. obtusa, the causal agent of apple black rot in the US, which produced large dark brown conidia.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.